Webster et al. Genome Medicine (2018) 10:64 https://doi.org/10.1186/s13073-018-0575-9 RESEARCH Open Access Increased DNA methylation variability in rheumatoid arthritis-discordant monozygotic twins Amy P. Webster1,2* , Darren Plant3, Simone Ecker2, Flore Zufferey4, Jordana T. Bell4, Andrew Feber2,5, Dirk S. Paul6, Stephan Beck2, Anne Barton1,3, Frances M. K. Williams4† and Jane Worthington1,3*† Abstract Background: Rheumatoid arthritis is a common autoimmune disorder influenced by both genetic and environmental factors. Epigenome-wide association studies can identify environmentally mediated epigenetic changes such as altered DNA methylation, which may also be influenced by genetic factors. To investigate possible contributions of DNA methylation to the aetiology of rheumatoid arthritis with minimum confounding genetic heterogeneity, we investigated genome-wide DNA methylation in disease-discordant monozygotic twin pairs. Methods: Genome-wide DNA methylation was assessed in 79 monozygotic twin pairs discordant for rheumatoid arthritis using the HumanMethylation450 BeadChip array (Illumina). Discordant twins were tested for both differential DNA methylation and methylation variability between rheumatoid arthritis and healthy twins. The methylation variability signature was then compared with methylation variants from studies of other autoimmune diseases and with an independent healthy population. Results: We have identified a differentially variable DNA methylation signature that suggests multiple stress response pathways may be involved in the aetiology of the disease. This methylation variability signature also highlighted potential epigenetic disruption of multiple RUNX3 transcription factor binding sites as being associated with disease development. Comparison with previously performed epigenome-wide association studies of rheumatoid arthritis and type 1 diabetes identified shared pathways for autoimmune disorders, suggesting that epigenetics plays a role in autoimmunity and offering the possibility of identifying new targets for intervention. Conclusions: Through genome-wide analysis of DNA methylation in disease-discordant monozygotic twins, we have identified a differentially variable DNA methylation signature, in the absence of differential methylation in rheumatoid arthritis. This finding supports the importance of epigenetic variability as an emerging component in autoimmune disorders. Keywords: Autoimmune disease, Rheumatoid arthritis, Epigenetics, DNA methylation, Twins Background hormone influences, infection, vitamin D intake and diet- Low disease concordance rates between monozygotic ary factors [2, 3], but few have been robustly confirmed. (MZ) twins (~ 15%) have revealed that environmental Epigenetics is the study of heritable modifications of exposures are important in rheumatoid arthritis (RA) DNA which can alter gene expression without changing [1]. Many putative environmental risk factors have been the DNA sequence and which can be influenced by en- investigated, including exposure to cigarette smoke, vironmental factors, such as smoking [4]. The most widely studied epigenetic phenomenon is DNA methyla- * Correspondence: [email protected]; [email protected] † tion, which may act as a composite measure of numer- Frances M. K. Williams and Jane Worthington contributed equally to this ous environmental exposures, making it an intriguing work. 1Arthritis Research UK Centre for Genetics and Genomics, Centre for candidate for investigation of diseases that involve both Musculoskeletal Research, The University of Manchester, Manchester, UK genetic and environmental factors, such as RA. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Webster et al. Genome Medicine (2018) 10:64 Page 2 of 12 Current evidence suggests that DNA methylation however, they have a difference in the range of DNA changes are associated with RA [5–10] but whether this methylation values between comparison groups. is due to intrinsic genetic differences, which can also in- We have examined genome-wide DNA methylation in fluence DNA methylation, is not yet known. Disease- both a DMP and DVP context using the Infinium discordant MZ twin pairs offer an ideal study design as HumanMethylation450 BeadChip array (Illumina) in they are matched for many factors, including genetic whole blood from 79 MZ twin pairs discordant for RA variation and as such they offer a crucial advantage in from two independent cohorts (Manchester and Twin- epigenetic studies [11]. Differences in methylation be- sUK, see Fig. 1). We identified a DVP signature in the tween MZ twins may capture the effects of environmen- absence of a DMP signature that suggest potential roles tally driven mechanisms, independent of genetically for multiple stress response pathways and potential epi- driven changes. Two small epigenome-wide association genetic disruption of RUNX3 transcription factor bind- studies (EWAS) of DNA methylation in MZ twins dis- ing sites in RA aetiology. We also identified shared cordant for RA have reported conflicting results. The DVPs in both RA and T1D, indicating potential shared first (n = 5 pairs) identifying no significant changes asso- pathways for autoimmune disorders. ciated with RA using the GoldenGate assay [12], while the second (n = 7 pairs) identified no significant differen- Methods tially methylated positions (DMPs), but one significantly TwinsUK participants differentially methylated region (DMR) using the Twin pairs discordant for RA were identified from the CHARM platform [13]. Due to the small sample sizes of TwinsUK register [19]. RA status was assessed through both studies, they were underpowered to detect subtle questionnaires between 1997 and 2002. In addition, an methylation differences [14] and have limited scope to advertisement was published in the National Rheuma- characterise the epigenomic landscape of RA-discordant toid Arthritis Society newsletter in spring 2013 to recruit twins. twin volunteers with RA. All MZ twins who answered To our knowledge, all studies of DNA methylation in positively were phone-interviewed by a rheumatology relation to RA have focussed on the identification of clinical fellow to confirm the diagnosis of RA based on DMPs or DMRs, which describe differential DNA the American College of Rheumatology 1987 criteria (n methylation levels at a particular CpG site or closely = 17 RA twins). In case of unclear diagnosis of RA, par- spaced group of CpG sites, respectively. In DMPs and ticipants were reviewed in clinic or were excluded. In DMRs, one group has a consistently higher level of addition, all patients willing to attend a visit were exam- DNA methylation than the comparison group (e.g. when ined clinically (n = 11 RA twins) by a clinical fellow comparing RA patients with healthy controls). Differen- under the supervision of a consultant rheumatologist. tially variable positions (DVPs) are another type of epi- Visits included detailed medical history, review of symp- genetic variation, the importance of which has recently toms, past and present medication (NSAIDS, disease- been elucidated in type 1 diabetes (T1D) and cervical modifying anti-rheumatic drugs (DMARDS) and/or bio- and breast cancer [15–18]. DVPs are CpG sites that do logical agents) and joint examination. Blood samples not necessarily have a large difference in mean DNA were collected from all subjects, from which DNA and methylation and therefore may not be classed as DMPs; serum were extracted and stored at − 80 °C. Fig. 1 Overview of study design. Rheumatoid arthritis-discordant twin pairs were recruited from the RA twins study in Manchester and TwinsUK in London, and genome-wide DNA methylation was investigated in the context of both differentially methylated positions and differentially variable positions Webster et al. Genome Medicine (2018) 10:64 Page 3 of 12 The healthy co-twins were also reviewed at the clinical reference-based Houseman method to infer relative pro- visit. Non-RA status was supported by both clinical and portions of cells [24]. Differences in cell composition be- immunological details, as all non-RA twins were sero- tween groups were tested using a Welch two-sample t negative, except one who was rheumatoid factor (RF) test. Additionally, we applied the recently developed EpiD- positive but clinically unaffected. ISH algorithm to infer cell composition, which confirmed the results obtained by Houseman’s algorithm (data not Manchester participants shown) [25]. Patients were selected from the Nationwide Rheumatoid Arthritis Twin Study based at the University of Manches- Identification of differentially methylated positions ter [1]. Twins were recruited in 1989 using a dual strategy: (DMPs) (1) all UK rheumatologists were contacted and requested A mixed effects model was used to test for DMPs from to ask all of their patients with RA whether they were a beta values using the CpGassoc